1. Field of the Invention
This invention relates to the modulation of gene expression, and to the modulation of mammalian gene expression regulated by methylation.
2. Summary of the Related Art
The modulation of expression of genes has long been pursued by researchers. For example, many mammalian diseases are associated with the over- or under-expression of certain genes. By modulating the expression of the gene whose aberrant expression is associated with a disease (or a predisposition to develop such a disease), the disease symptoms may be alleviated.
For example, it would be desirable to modulate the enzyme, DNA Methyltransferase (DNA MeTase), which mediates the methylation of mammalian DNA.
Methylation of mammalian DNA is enzymatically mediated by the covalent modification of the fifth carbon position of the pyrimidine ring of cytosine in CpG dinucleotides. Changes in the pattern of DNA methylation have been correlated with a number of processes in eukaryotes. Holliday (1990) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 326: 329-338 discusses the role of methylation in parental imprinting. Antequera et al. (1989) Cell 58: 509-517 discusses the significance of methylation in developmental regulation. Fedoroff et al. (1989) Cell 56: 181-191 discloses that methylation is involved in transposition. Hare and Taylor (1985) Proc. Natl. Acad. Sci. USA 82: 7350-7354 discloses that methylation is also implicated in DNA repair. In addition, Gartler and Riggs (1983) Ann. Rev. Genet. 17. 155-190 correlates methylation with X chromosome inactivation, while Bird et al. (1986) Nature 321: 209-213 discloses that methylation also plays a pivotal role in chromatin organization.
While several observations have suggested a role for DNA methylation in cancer pathogenesis, there has been a great deal of disagreement as to the mechanisms involved. Szyf et al. (1996) Pharmacol. Ther. 70 (1): 1-37 discloses that methylation and demethylation activities are critical components of some tumorigenic growth forms. MacLeod and Szyf (1995) J. Biol. Chem. 270: 8037-8043 discloses that the level of DNA methyltransferase activity may be a nodal control point over oncogenic growth. On the other hand, others have dismissed the theory that DNA Methyltransferase plays a causal role in oncogenesis.
A significant obstacle to the investigation of the role of methylation lies in the deficiencies of available methods to modulate methylation itself. To date, most studies have relied on 5-aza-C and/or 5aza-dC to inhibit DNA methylation by forming a stable adduct with DNA methyltransferase, thus mimicking the transient covalent intermediate complex believed to be formed during methylation (see, e.g., Wu and Santi (1987) J. Biol. Chem. 262: 4778-4786). This approach, albeit effective in reducing DNA methyltransferase activity and correlating with tumorigenesis inhibition, is therapeutically deficient. Unfortunately, threshold concentrations of 5-azaC or 5-aza-dC empirically necessary to inhibit DNA methyltransferase activity have been found to be toxic to mammals (juttermann et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11797-11801; Laird (1997) Mol. Med. Today 3: 223-229).
More recently, Szyf et al. (1995) J. Biol. Chem. 267: 12831-12836, has disclosed a more promising approach to the modulation of DNA methylation using expression of antisense RNA complementary to the DNA methyltransferase mRNA to study the effect of methylation on cancer cells. U.S. Pat. No. 5,578,716, discloses the use of antisense oligonucleotides complementary to the DNA methyltransferase gene to inhibit gene expression. These developments have provided powerful new tools for probing the role of methylation in numerous cellular processes. In addition, they have provided promising new approaches for developing therapeutic compounds that can modulate methylation levels.
The effect of antisense inhibition is not immediate, due to the half-life of the DNA methyltransferase enzyme. Thus, although the expression of DNA methyltransferase is modulated, residual enzyme can continue to methylate DNA until such enzyme is degraded. Furthermore, polysome-associated DNA methyltransferase mRNA may also persist for some time, allowing additional translation to produce additional enzyme. In addition, the pharmacodynamic properties of oligonucleotides suggest that lower doses than are currently used could be beneficial (Agrawal et al. (1995) Clinical Pharmacokinetics 28: 7-16; Zhang et al. (1995) Clinical Pharmacology and Therapeutics 58: 44-53).
Therefore, there remains a need to develop more effective methods for the modulation of the expression of genes, such as DNA methyltransferase, that would reduce the required dosage of antisense oligonucleotides, specific inhibitors of gene products, or other agents currently used while at the same time effectively accomplish the inhibition of gene expression.